An optimized protocol to assess SUMOylation in the plant Capsella rubella using two-component DEX-inducible transformants

Summary Here, we present an efficient protocol to test the SUMOylation of a target protein in the plant Capsella rubella based on overexpression of dexamethasone (DEX)-inducible tagged proteins. We describe the construction of two-component, FLAG-tagged DEX-inducible plasmids. We then detail the transformation of Capsella, followed by DEX treatment and SUMOylation assays. This protocol can be widely applied to proteins with expression restricted to specific cells and tissues using native promoters as well as proteins whose overexpression leads to embryo lethality. For complete details on the use and execution of this profile, please refer to Dong et al. (2020).


SUMMARY
Here, we present an efficient protocol to test the SUMOylation of a target protein in the plant Capsella rubella based on overexpression of dexamethasone (DEX)-inducible tagged proteins. We describe the construction of two-component, FLAG-tagged DEX-inducible plasmids. We then detail the transformation of Capsella, followed by DEX treatment and SUMOylation assays. This protocol can be widely applied to proteins with expression restricted to specific cells and tissues using native promoters as well as proteins whose overexpression leads to embryo lethality. For complete details on the use and execution of this profile, please refer to Dong et al. (2020).

BEFORE YOU BEGIN
Conjugation of small ubiquitin-related modifier (SUMO) to the proteins provides the plants with a fast and dynamic regulatory mechanism to cope with environmental fluctuations and intrinsic developmental clues (Mukhopadhyay and Dasso, 2007). Similar to ubiquitin, SUMO becomes covalently attached to lysyl e-amino groups in the target proteins by the action of a dedicated enzymatic cascade (Miura and Hasegawa, 2010). Upon conjugation, SUMOylation usher in profound impact on the target protein, including the way they interact with other proteins and nucleic acids, stability and subcellular localization (Mukhopadhyay and Dasso, 2007). Interestingly, SUMOylation is a highly dynamic and reversible process as the SUMOs can be trimmed off by specific cysteine proteases in developmental-context dependent manner (Miura and Hasegawa, 2010). All these features make detection of SUMOylation a challenge in analyzing the biochemical property of a protein of interest.
Note: There is no special requirement for the E. coli competent cell, we recommend the DH5alpha cell strain because of the high efficiency. Higher ratio between the plasmid and E. coli competent cells will result in lower transformation rate.
6. Pick 2-4 white colonies (against blue ones) and extract the plasmids by Midi-Prep and verify the insertion by sequencing.
CRITICAL: Always prepare the plasmid by Midi-Prep as it is critical for multiple DNA assemblies in the following step.  (for reaction and program set up, see Figures  1B and 1D, and Table S2). 8. Transform the reaction product into DH5-alpha competent Escherichia coli cells in a 1:10 ratio and put 10-20 mL on LB plate containing 100 mg/L Carbenicillin, 25 mg/L IPTG and 40 mg/L X-gal, grow the bacterial at 37 C for 14 h.

PCR products
Note: Higher ratio between the plasmid and E. coli competent cells will result in lower transformation rate.
9. Pick 2-4 white colonies (against blue ones) and extract the plasmids by Midi-Prep and verify the insertion by sequencing. 10. Golden-Gate L2 reaction using BpiI and T4 ligase (for reaction and program set up, see Figures  1C and 1D, and Table S2). 11. Transform the reaction product into DH5-alpha competent Escherichia coli cells in a 1:10 ratio and put 20-30 mL on LB plate containing 50 mg/L Kanamycin, grow the bacterial at 37 C for 14 h.
Note: Higher ratio between the plasmid and E. coli competent cells will result in lower transformation rate.
12. Pick 1-2 white colonies (against orange ones) and extract the plasmids and verify the insertion by sequencing.

Prepare the two-component DEX-inducible Capsella transformants
Timing: $3-4 months In the following section, we describe the detailed construction process of the two component DEXinducible system (pLhGR>>) for the CrIND protein in the Capsella htb-1 mutant, which harbors a loss-of-function mutation in the SUMO protease (Dong et al., 2020). For the mutant CrIND protein, full length CrIND coding sequence with lysine at position 124 (K124) mutated to arginine (R) was amplified by recombination PCR. We used the above mentioned Golden-gate cloning protocol to generate the pLhGR>>CrIND K124R :33FLAG plasmids. The transformation protocol of Capsella followed the floral dipping method described in Dong et al. (2019).

Preparation of Capsella plants for floral dipping
Timing: $35-40 days a. Wash the seeds once with sterilized ddH 2 O, centrifuge transiently to collect the seeds. b. Remove the water completely and resuspend the seeds with 5% sodium hypochlorite supplied with 0.1% Triton X-100 for 3 min, remove the sodium hypochlorite completely and wash the seeds four times with sterilized ddH 2 O. c. Microwave the seed growth MS medium, cool down the medium in water to $45 C add 20 mL 100 mM GA 3 to a final contraction of 10 mM. Mix gently and pour the medium into the square petri dishes (10 cm) in the laminar hood.
Note: Gibberellin (GA 3 ) is required to the break the seed dormancy in Capsella.
CRITICAL: Always add the GA 3 when the medium cooling down to $45 C as GA 3 is sensitive to high temperature.
d. Put the sterilized seeds on MS agar plates evenly (Figure 2A), and store the seeds in the dark at 4 C for 24-48 h for stratification.

OPEN ACCESS
CRITICAL: Stratification at 4 C for 24-48 h is required to generate unified seed germination.
e. Transfer the seeds into the growth room under long-day (16 h light/8 h dark) conditions at 22 C for 12 days, the seeds are normally germinated in $24-48 h. f. The 10-day-old seedlings were transplanted to soil to grow in the growth room under longday (16 h light/8 h dark) conditions at 22 C ( Figure 2B).

Transformation of Capsella
Timing: $14-21 days a. Transform the pLhGR>>CrIND K124R :33FLAG plasmids into the Agrobacterium tumefaciens strain LBA4404 by cold shock method, and select the colonies on YEB medium containing 50 mg/L Kanamycin, 100 mg/L Streptomycin and 50 mg/L Rifampicin at 28 C in dark.
Note: The LBA4404 strain grows slower in YEB medium and the activity is much higher.
CRITICAL: LBA4404 strain is required for Capsella transformation as it increases the transformation efficiency dramatically compared with other strains, such as GV3101.
b. Pick $5-6 colonies and mix them in 20 mL liquid YEB medium containing 50 mg/L Kanamycin, 100 mg/L Streptomycin and 50 mg/L Rifampicin at 28 C, 200 rpm in a growth chamber. c. Culture the Agrobacterium at 28 C for 18-24 h to 1.8-2.0 (OD 600 ) and collect the bacteria by centrifugation at $4,500 rcf (g) for 10 min at 25 C. d. Resuspend the bacteria using 5% sucrose in sterilized ddH 2 O supplied with 0.03% Silwet L-77 to OD 600 of 0.8-0.9.
CRITICAL: The OD 600 value and concentration of Silwet L-77 is key to the transformation rate in Capsella. Please do use the recommended value indicated in the above step.
e. Subject htb-1 Capsella plants that start to bolt to the first dipping ( Figure 2C), and subsequently, leave in the dark for $20-24 h at 22 C.
Note: We placed the dipped plants in a black plastic bag for keeping dark and humidity ($95%), this process is crucial for a successful transformation experiment.
f. Repeat the floral dipping twice ( Figure 2D), with at least seven days intervals between each dip. g. After finishing the floral dip, place the plants in the growth room under long-day (16 h light/ 8 h dark) conditions at 22 C until fruits and seeds are mature. h. Harvest and desiccate the seeds at 37 C for $3-5 days, then keep seeds in a dry environment at 4 C. i. Sterilize the seeds following the process in the aforementioned steps. step 13a and 13b. j. Screen the transformants on selection MS medium containing 25 mg/L DL-phosphinothricin, 200 mg/L Cefotaxime and 10 mM GA 3 . Normally, $5-10 positive transformants will be obtained on each plate ( Figures 2E and 2F).
Note: Cefotaxime is required to suppress the Agrobacterium on the seed coat, lower concentration will result in overgrowth of the Agrobacterium, which in turn affects the selection process.
Note: For Hygromycin selection, 40 mg/L Hygromycin is recommended on the selection plate.
k. The transformants should be transplanted and selfed to generate enough T 2 plants for further experiment.

MATERIALS AND EQUIPMENT
Essential equipment: Low-temperature centrifugate, large space fridge or cold room, 3D gyratory rocker, orbital shaker, protein electrophoresis system, vacuum concentrator (minimum to 100 mbar), magnetic rack and thermostatic bath (25 C-105 C).

Antibiotics preparation Prepare Kanamycin solution
Dissolve 100 mg Kanamycin powder in 2 mL ddH 2 O. Sterilize using 0.22 mm filters in a laminar hood to a final concentration of 50 mg/mL. Aliquot (500 mL for each) and store at À20 C up to six months.

Prepare Carbenicillin solution
Dissolve 200 mg Carbenicillin powder in 2 mL ddH 2 O to a final concentration of 100 mg/mL. Sterilize using 0.22 mm filters in a laminar hood. Aliquot (500 mL for each) and store at À20 C up to six months.

Prepare Spectinomycin solution
Dissolve 200 mg Spectinomycin powder in 2 mL ddH 2 O to a final concentration of 100 mg/mL. Sterilize using 0.22 mm filters in a laminar hood. Aliquot (500 mL for each) and store at À20 C up to six months.

REAGENT or RESOURCE SOURCE IDENTIFIER
X-gal solution (20 mg/mL) Biosharp Cat#BL546A Dissolve 200 mg Streptomycin powder in 2 mL ddH 2 O to a final concentration of 100 mg/mL. Sterilize using 0.22 mm filters in a laminar hood. Aliquot (500 mL for each) and store at À20 C.
Prepare Cefotaxime solution Dissolve 1.0 g Cefotaxime powder in 5 mL ddH 2 O to a final concentration of 200 mg/mL. Sterilize using 0.22 mm filters in a laminar hood. Aliquot (500 mL for each) and store at À20 C up to six months.
Plant growth related reagents Prepare seedling growth MS medium Dissolve 34.6 mg Gibberellic acid (GA 3 ) in 1 mL ethanol in a laminar hood. Store at À20 C up to six months.
Prepare 10 mM Dexamethasone (DEX) Dissolve 4 mg DEX in 1.0 mL ethanol in a laminar hood. Store at À20 C up to two months.
Prepare 20% Triton X-100 Dilute 1 mL 100% Triton X-100 with 4 mL sterilized ddH 2 O to 5 mL 20% Triton X-100. Store at room temperature (25 C) up to six months. Prepare 1 M Dithiothreitol (DTT) Dissolve 1.543 g DTT in 10 mL of sterilized ddH 2 O. Make 1.0 mL aliquots and store at À20 C up to six months.

Reagent Final concentration Amount
Prepare 100 mM Phenylmethylsulfonyl fluoride (PMSF) Dissolve 0.174 g PMSF in 10 mL dimethyl sulfoxide (DMSO). Make 1.0 mL aliquots and store at À20 C up to six months.
Note: Due to toxicity of PMSF and DMSO, the PMSF solution should be prepared in a fume hood.

Prepare 1003 Complete Protease Inhibitor Cocktail
Dissolve 1 tablet in 500 mL sterilized ddH 2 O. Store at À20 C up to two months.
Prepare 2 M N-Ethylmaleimide (NEM) Dissolve 0.25 g NEM in 1.0 mL ethanol. Store at À20 C up to two months.
Note: NEM is toxic and an irritant, wear PPEs and conduct this step in a fume hood.

Prepare protein extraction buffer
For 10 mL protein extraction buffer, mix 100 mL 1 M DTT, 100 mL 100 mM PMSF, 100 mL 1003 Complete Protease Inhibitor Cocktail, 100 mL 2 M NEM, and fill up to 10 mL with GTEN buffer. Note: The protein extraction buffer should be fresh-prepared and pre-cooled on ice before use.
CRITICAL: NEM is required in the protein extraction buffer as it dramatically inhibit SUMO protease activity in the cell lysate.

Prepare 10 mL IP wash buffer
For 10 mL IP wash buffer, mixed 1 mL 1 M DTT, 100 mL 100 mM PMSF, 100 mL 1003 Protease Inhibitor, and fill up to 10 mL with GTEN buffer. Store at 4 C for 12 h.
Western-blot related reagents Prepare 1.5 M Tris-HCl pH 8.8 Dissolve 181.5 g Tris in 800 mL ddH 2 O, adjust pH to 8.8 with HCl, and fill up to 1 L with ddH 2 O. Autoclave at 121 C for 20 min. Store at room temperature (25 C) up to six months.
Prepare 1.0 M Tris-HCl pH 6.8 Dissolve 121.0 g Tris in 800 mL ddH 2 O, adjust pH to 6.8 with HCl, and fill up to 1 L with ddH 2 O. Autoclave at 121 C for 20 min. Store at room temperature (25 C) up to six months.

Note:
The 10% protein separation gel should be fresh-prepared. Prepare 5% non-fat milk blocking buffer Dissolve 2.5 g Blotting-Grade Blocker in 50 mL 13 TBST buffer, mix gently and store at 4 C up to 48 h.

STEP-BY-STEP METHOD DETAILS SUMOylation test: Prepare seedlings and DEX treatment
Timing: $12-13 days In the following section, we will describe on how to prepare the plant material for the protein extraction.
1. Sterilize the T2 seeds of pLhGR>>CrIND K124R :33FLAG using the aforementioned methods. 2. Screen the seeds on selection MS medium containing 25 mg/L DL-phosphinothricin, 200 mg/L Cefotaxime and 10 mM GA 3 . 3. Put the seeds evenly on the medium and store the seeds in dark at 4 C for 24-48 h for stratification. 4. Transfer the seeds into the growth room under long-day (16 h light/8 h dark) conditions at 22 C for 12 days, the seeds are normally germinated in $24-48 h ( Figure 3A). 5. Put 100 mL 10 mM DEX into 100 ml DEX treatment MS liquid medium. Set a mock experiment by putting 100 mL 100% ethanol into the DEX treatment MS medium. 6. Collect the 10-day-old seedlings ($500 individuals per treatment) and put them into the DEX MS liquid medium and DEX mock MS liquid medium, respectively ( Figure 3B). Fix the flasks on the shaker, grow the seedlings at 80 rpm in the growth room under long-day (16 h light/8 h dark) conditions at 22 C for $12-16 h.

SUMOylation test: Total protein extraction from seedlings
Timing: $2.5 h In the following section, we will describe the details on how to extract the total proteins from the DEX-induced seedlings. Note: Fixative must be freshly prepared. Seal the fixative after the preparation since formaldehyde is toxic and evaporative.
8. Harvest the DEX-treated seedlings, wrap the sample in a single layer of miracloth and cross-link the proteins in the fixing buffer using 50 mL beaker under vacuum (minimum to 100 mbar) for 15 min. Release the vacuum slowly to avoid any damage to plant tissues.
Note: As the SUMOylation is a reversible and highly dynamic process in the cell, we recommend fix the proteins using 1% formaldehyde under vacuum for 15 min.
9. Stop the fixing process by adding 6.25 mL 2 M Glycine to a final concentration of 125 mM under vacuum for another 5 min. 10. Rinse the sample 3 times with ddH 2 O, dry the sample with lab tissues and weigh the sample.
Note: As paraformaldehyde is toxic and evaporative, wear a mask and change the buffers in the fume hood.
11. Pre-cool the mortar, pestle, 15 mL falcon tubes and spatula in liquid nitrogen until they are completely cooled down. 12. Grind the sample ($1.0 g) in liquid nitrogen $2-3 times using mortar and pestle into fine powder ( Figure 3C). CRITICAL: Grind the sample as much as you can, the finer the powder more protein will be extracted.
13. Transfer the sample powder to the cooled conical tube (15 mL) using the pre-cooled spatula. 14. Add 2.53V (2.5 mL) protein extraction buffer into the samples.
CRITICAL: NEM in the protein extraction buffer is absolutely necessary for a successful SUMOylation test as it dramatically inhibits the SUMO protease activity.
15. Gently invert the tube to mix the sample powder with the protein extraction buffer. 16. Fix the tubes on the rotator and extract the protein at a speed of 40 rpm for 1 h at 4 C (Figure 3D). 17. Pre-cool the centrifuge at 4 C when extracting the proteins. 18. Centrifugate the samples at ～15,000 rcf (g) and 4 C for 10 min. 19. Collect the supernatant and split it into two 2.0 mL eppendorf tubes ($1.5 mL/each) on ice. 20. Centrifugate the samples again at ～15,000 rcf (g) and 4 C for 5 min. 21. Transfer the supernatant to new 2.0 mL eppendorf tube on ice.
Note: Repeat step 19 and 20 until the supernatant is clear.
CRITICAL: Always perform step 14-21 on ice as low temperature is critical to protect the proteins from degradation.
22. Take 50 mL of lysate as input and add 50 mL of 23 SDS loading buffer, boil the sample at 100 C for 5 min.
Note: Open the tube lid carefully after boiling.
23. Store the input samples at À20 C.

SUMOylation test: Immunoprecipitation with anti-FLAG M2 beads
Timing: $4 h In this section, we will introduce the details of recombinant protein immunoprecipitation using anti-FLAG M2 magnetic beads.
24. Wash the anti-FLAG M2 magnetic beads (20 mL/g sample) $3 times with 1 mL IP wash buffer. For each wash, rotate the samples with a speed of 40 rpm for 5 min at 4 C.
Note: Mix the anti-FLAG M2 magnetic beads gently before use. A magnetic rack is required for collecting the beads after each wash. Between each wash, please keep the samples on the magnetic rack for $2-3 min at 4 C to allow complete collection of the beads ( Figure 3E).
25. Resuspend the anti-FLAG M2 magnetic beads with 50 mL IP wash buffer per tube. Add 50 mL beads into the lysate of each eppendorf tube, mix the samples on a rotator with a speed of 40 rpm for $2-3 h at 4 C. 26. Collect the anti-FLAG M2 magnetic beads with the magnetic rack. Discard the lysate and wash the beads $4 times using 1 mL IP washing buffer. For each wash, keep rotating the samples at a speed of 40 rpm at 4 C for 5 min.
Note: Between each wash, please keep the samples on the magnetic for $2-3 min at 4 C to allow complete collection of the beads.

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27. Add 50 mL of 23 SDS loading buffer, boil the beads at 100 C for 10 min, mix the sample from the same genotype, a total of 100 mL IP protein are collected. 28. Store the IP samples at À20 C.
SUMOylation test: SDS-PAGE separation of proteins and membrane transfer Timing: $3.5 h In this section, we are going to introduce the detailed protocol on how to separate the proteins and transfer the protein onto the PVDF membrane for western-blot.
29. Put two SDS-PAGE gels (each SDS-PAGE contains a 5% stacking gel and a 10% separation gel) in the protein electrophoresis system.

Note:
The SDS-PAGE gels should be prepared in fresh; we don't recommend using commercial pre-made SDS-PAGE gels for this SUMOylation test protocol.
30. Load 10 mL IP protein sample, 5 mL input sample and 2.5 mL protein marker into the SDS-PAGE gels. 31. SDS-PAGE electrophoresis at the voltage of 90 V for 1.5 h at room temperature.

Note:
The 13 SDS running buffer should be fresh-prepared and single use.
32. Cut the PVDF membrane and filter pater into 6.0 cm 3 8.5 cm in size, rinse the membrane with 100% methanol for 2 min in a square petri dish, then gently remove the methanol and decant 13 transfer buffer (pre-cooled) in the petri dish. 33. Carefully collect the SDS-PAGE gels and put them into 13 transfer buffer (pre-cooled) in a square petri dish. 34. Soak sponge pad, filter paper in the 13 transfer buffer (pre-cooled). 35. Make the gel-membrane transfer sandwich in the clump (negative charge-sponge pad-filter paper-SDS-PAGE gel-PVDF membrane-filter paper-sponge pad-positive charge).
Note: Care should be taken when putting the PVDF membrane onto the SDS-PAGE gel.
CRITICAL: Avoid bubbles between the SDS-PAGE gel and PVDF membrane. The bubbles could be avoided by flushing the gel with 13 transfer buffer before adding the PVDF membrane. Also, be sure that the gel and membrane are in order with the charge.
36. Transfer the proteins onto the PVDF membrane by electrophoresis at the voltage of 100 V for $1.5-2 h at 4 C.
CRITICAL: Be sure that the transfer buffer is always in cold environment. Carry out this process in the cold room or in the fridge. Also, a pre-cooled (À20 C) ice rack is recommended to put into the electrophoresis tank.

SUMOylation test: Western blot
Timing: $16 h In the following section, we will provide the details on how to detect the protein SUMOylation using anti-SUMO1 antibodies by western blot.
37. Collect the membrane in small WB incubation boxes and decant 10 mL 5% non-fat milk blocking buffer in each box, keep on the shaker at 4 C for 2 h ( Figure 3G).
Note: Check the color of the biggest protein marker on the membrane as an indication of protein transfer efficiency.
CRITICAL: Blocking for 2 h at 4 C is enough to reduce the unspecific backgrounds.
38. Remove the blocking buffer and add another 5 mL 5% non-fat milk blocking buffer. Dilute the antibody in the blocking buffer, 1:1,000 for anti-SUMO1 and 1:5,000 for anti-FLAG-HRP. Keep on the shaker at 4 C for $12-15 h.
Note: anti-FLAG antibody could also be used in the step, the concentration, incubation time, temperature and secondary antibodies can be adjusted following the instructions of the anit-FLAG antibody.
CRITICAL: Aliquot the antibodies upon its arrival, keep the aliquots at À20 C and avoid repeated freezing and thawing.
39. Remove the primary antibody solution completely and rinse the membrane with the 13 TBST buffer three times, with 5 min each time on the shaker at 4 C.
Note: The primary antibody solution can be reused in $3-4 times and keep in À20 C.
CRITICAL: Remove the primary antibody completely after each wash using a pipette, residual primary antibody on the membrane will generate high back-ground signals.
40. Decant 5 mL secondary antibody (1:10,000 anti-rabbit IgG in 5% non-fat milk blocking buffer) into the WB incubation box. Keep on the shaker at 4 C for 2 h Note: The anti-FLAG antibody has already conjugated with HRP, this step can be passed for the anti-FLAG membrane and directly go to step 39.
41. Remove the secondary antibody solution completely and rinse the membrane with the 13 TBST buffer three times, 5 min each time on the shaker at 4 C.
Note: The secondary antibody solution can be reused in $5-6 times and keep in À20 C.
CRITICAL: Remove the secondary antibody completely after each wash using a pipette, residual secondary antibody on the membrane will generate high back-ground signals.
Note: For the anti-FLAG membrane, the explosion time normally takes $1 min to produce a desirable signal. For the anti-SUMO1 membrane, the input sample normally need $30 s to 1 min to generate the signal and the IP sample may take longer ($15 min).

EXPECTED OUTCOMES
Generating Capella transformants By using the optimized Capsella transformation protocol described in this paper, a number of positive transformants will be observed on the plates containing 25 mg/L DL-phosphinothricin (Basta) or 40 mg/L Hygromycin antibiotics. The overall transformation rate is around $0.15%, which is close to ll OPEN ACCESS the rate described in Arabidopsis (Clough and Bent, 1998). In addition, the methods of plant transformation described in this paper is probably applicable to other Brassicaceae species as well.
SUMOylation test using the two-component DEX-inducible system The CrIND proteins will be induced significantly upon DEX treatment of the seedlings ( Figure 3H). SUMOylation of the CrIND will be observed from the WB of purified proteins using anti-FLAG beads and anti-SUMO1 antibodies ( Figure 3H). It should be noted that the specificity of SUMOylation has to be verified by doing a parallel experiment using a mutant protein with modified SUMOylation site (K124R) and possibly access functional relevance of the SUMOylation residual in the respective mutant background ( Figure 3H).

Advantages and potential applications
One general characteristic of SUMOylation lies in that its dynamic and reversable modification nature on the lysine residual of the target protein in that the protein size change that can be recognized by SUMO antibodies (Mukhopadhyay and Dasso, 2007). Compared with in vitro SUMOylation test system in E. coli and in vivo test system in tobacco leaves (Kong et al., 2017;Qu et al., 2020), which is based on the co-overexpression of the SUMOs and key components in the SUMOylation pathway, the use of DEX-inducible overexpression system provide more reliable and stable results as it using the native SUMOylation system in planta.
As SUMOylation process shared a lot of characteristics to that of ubiquitinoylation (Miura and Hasegawa, 2010), the experimental pipeline described herein could also be modified to test other kind of post-translational modifications, such as ubiquitinoylation and methylation, acetylation, and so on.

LIMITATIONS
As stable transformants are required to generate enough plant material for the DEX-treatment and downstream experiment, it may take longer time ($4-5 months) to finish this protocol than in vitro SUMOylation test in E. coli (Kong et al., 2017;Qu et al., 2020). This protocol has the same constraints as any standard transgenic approach, in that the plant species or variety must be efficiently transformable to generate sufficient independent transformants. However, protoplast-based transient expression system could be an alternative approach for species that are recalcitrant to transformation (Zhang et al., 2011).
As SUMOylation is a fast and reversible process in the cells by the action of SUMO protease. Therefore, a SUMO protease mutant may be required before hand to generate stable results.

Potential solutions
Check the insertion sequence to see is there any BpiI and BsaI restriction site, any unexpected restriction sites will disrupt the ligation outcomes.
Always purified the PCR products from the agarose gel.
Check the plasmid extraction protocol see if the plasmids are prepared by midi-prep.
Re-calculate the relative amount (ng) of individual plasmids needed for each reaction. Each part/ module has to be 2:1 ratio relative to the acceptor plasmids. For a simple calculation, please fill in your plasmid concentration in Table S2, the amount and volume will be automatically calculated.
It is possible that the concentration of antibodies is too low or the duration of antibody incubation is not adequate. The concentration and incubation time of antibodies should be adjusted for different proteins and plant species.
The antibodies should be aliquoted upon arrival to avoid degradation with repeated freezing and thawing.
SUMOylation is a reversable and dynamic post-translational modification controlled by SUMO proteases (Augustine and Vierstra, 2018). The SUMO protease could be suppressed by NEM, which is very evaporative in ethanol. Check the concentration of NEM added into the protein extraction buffer.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Lars Østergaard (lars.ostergaard@jic.ac.uk).

Materials availability
The Capsella lines and plasmids associated with this protocol are available upon request.

Data and code availability
This study did not generate unique datasets or code.